xylene or paraffin.
However, they both greatly alter the
STAINING OF LIPIDS
- Lipid refers to all fat and fat like, or fat containing
substances. This includes triglycerides, fatty acids,
lipoproteins and glycolipids.
- generally classified into simple lipids, compound lipids,
and derived lipids.
- They all have a common property of solubility in
organic solvents and insolubility in water.
1. Simple lipids (neutral fat): esters of fatty acids with
alcohols and are usually found in the body as energy
stores in adipose tissue. Triglycerides are esters of
fatty acids with glycerol constituents, serving as
storage fats in animals with high solubility for
certain non-ionic colored substances (lysochromes)
stainable by Sudan Black B, Sudan IV and Oil Red
0.
2. Compound lipids: consist of a fatty acid, an
alcohol and one or more other groups such as
phosphorus or nitrogen. They are generally found in
the central nervous system.
a. Phospholipids are the important components of
cellular membranes particularly found in
mitochondria and nervous tissue elements and
are readily stained by Sudan Black B and acid
hematin.
b. Glycolipids are composed of fatty acids and
hexoses, possessing characteristics of both
lipids and carbohydrates and are therefore
stained by Sudan Black B and PAS techniques.
3. Derived lipids: fatty acids that are derived from
hydrolysis of simple and compound lipids.
Examples are cholesterol, bile acids, sex hormones
and adrenocortical hormones.
Adipose Tissue (Fat)
- distributed throughout the body in distinct “white” and
“brown” adipose tissue depots.
- White adipose tissue (WAT) is largely composed of
unilocular lipid-filled adipocytes that specialize in lipid
storage, whereas brown adipose tissue (BAT) is largely
composed of multilocular adipocytes that specialize in
lipid burning.
- Fat cells appear as “signet rings” on H&E stain
because large lipid droplet displace the nucleus and
remainder of the cytoplasm to the edge of the cell.
- Oxidation of phospholipids by chromate fixation
renders them non-extractable by alcohol, toluene,
chemical reactivity of the lipids, which can adversely
affect staining.
- Formol-calcium: fixative of choice for lipid
histochemistry, and is prepared by adding 2% calcium
acetate to 10% formalin.
- Lipid bodies, also named lipid droplets or
adiposomes, are distributed in the cytoplasm as roughly
spherical organelles lacking a delimiting classical
bilayer membrane, but surrounded by an outer
monolayer of phospholipids, which at least in some cells
may have a unique fatty acid composition.
- Cytoplasmic lipid droplets are neutral lipids, usually
triacylglycerols or cholesteryl esters. Because lipid
bodies can be destroyed by drying or fixation and
staining with alcohol-based reagents, there are
consequently some methodological limitations to their
study.
- Lipids are difficult to demonstrate histologically
because they dissolve in the solvents used for paraffin
processing and, to a lesser degree, in celloidin
processing.
- Frozen sections may be required in order to stain for
lipids.
- Triglycerides are always completely removed in
properly cleared and paraffin infiltrated blocks
- Lipids bound to other materials, such as lipoproteins,
lipofuscins and myelin may not be completely removed
by solvents used during paraffin processing and
significant amounts may still be present in the tissue
sections.
- Lipids are best demonstrated on cryostat sections of
fresh unfixed tissue since there is no really good fixative
available.
- Formalin only preserves those lipids that are already
more or less firmly bound to proteins (such as
lipofuscins and granules of leukocytes).
Lipochrome (lipofuscin) pigments
- the breakdown products within cells from oxidation of
lipids and lipoproteins.
- They are the wear-and-tear pigments found most
commonly in heart, liver, CNS, and adrenal cortex.
- PAS positive and variably acid fast. They stain with
Ziehl-Neelsen.
- Sudan black B and Sudan Red positive.
- can be demonstrated by Schmorl's method which also
stains for melanin.
- may also exhibit a strong orange auto fluorescence in
formalin-fixed, unstained paraffin sections.

- Lipids present in fat embolism, fatty liver and atheroma
may be fixed for staining in paraffin sections by
exposing the sections to an emulsion of linoleic acid and
lecithin in 70% ethylene glycol at 56oC for 3 days.
- These tissues are then treated with 2% chromic acid at
4°C for 24 h followed by 24h in 5% sodium bicarbonate,
with appropriate rinsing between solutions.
- Paraffin sections of these tissues then stained with a
lipid-soluble dye such as Oil Red O.
- The demonstration of fat embolism with good quality
tissue detail is made practical by the method, which is
convenient and inexpensive.
- Phospholipids and neutral fats will be lost during routine
dehydration and embedding unless they are treated with
potassium dichromate or osmic acid, which are the only
agents that truly fix lipids.
- Oxidation of phospholipids by chromate fixation
renders them non-extractable by alcohol, toluene,
xylene or paraffin.
- Formol-calcium: fixative of choice for lipid
histochemistry, and is prepared by adding 2% calcium
acetate to 10% formalin.
- To preserve the lipids, polyethylene glycols
(carbowaxes) are sometimes used in lieu of the usual
dehydration and embedding process. However, this
technique is not widely utilized, and in most centers,
neutral fats are still best demonstrated in frozen sections
of fixed or unfixed tissue.
- Formol-calcium fixed tissue blocks are also suitable for
cryostat sections, and should be mounted on chrome-
gelatin coated slides, because fixation causes the
endogenous tissue proteins to lose their adhesive
properties.
- histochemical techniques are the common methods of
choice for demonstrating lipids in tissue sections.
Fat Stains and Sudan Dyes
- Sudanophilia: property of tissues to be stained with fat
or oil-soluble dyes, regardless of the type of dye used,
due to their essential lipid nature.
- Staining with these dyes is regarded as specific for
lipids, especially for simple lipids (neutral fat).
- Oil soluble dyes are usually divided into the main
groups:
1. Basic Aryl amines with very low water solubility:
a. Sudan Black B - most sensitive lipid stain
known
b. Sudan Red VII B
2. B-Naphthols such as the original diazo dyes
a. Sudan III (C.I. No. 26100)
b. Sudan IV (Scharlach B) C.I. No. 26105 -
staining fats with a more brilliant or deeper red
color than Sudan III which stains lipids orange-
red
- Sudan dyes are a group of lipid soluble solvent dyes
often called lysochromes.
- Sudan III was the first of these dyes to be introduced
in 1896, followed by Sudan IV (Scharlach R) in 1901.
- Sudan III: predominantly used for staining
triglycerides in animal tissues (frozen sections).
- also used to stain some protein bound lipids in paraffin
sections.
- The most sensitive and versatile: Sudan Black B,
which was introduced in 1935.
o it stains phospholipids as well as neutral fats
o does not stain crystalline cholesterol, and free fatty
acids tend to be dissolved in the alcoholic dye bath.
- For frozen sections, cut tissues about 15 micra thick are
usually stained with.
- Scharlach R or with Oil Red 0: stains neutral fats and
lipofuscin well.
Oil Red O
- used to demonstrate the presence of fat or lipids in fresh,
frozen tissue sections.
- a fat-soluble diazo dye, and is classified as one of the
Sudan dyes which have been in use since the late 1800s.
- not a true special stain, since it can’t form bonds with
lipid components.
- a pigment that functions as an oil-soluble colorant, and
the technique represents a physical method of staining.
- For general use, 70% alcohol is an adequate solvent for
Oil Red 0 and Sudan black. Containers should always
be kept covered except when the tissue is being placed
into and taken out of solution to prevent evaporation,
particularly of their alcoholic component.
Sudan Black Method for Lipids
Fixation: Formaldehyde calcium with post-chroming
Section: Unfixed cryostat sections preferred, or frozen
sections post-fixed in formol calcium
Method:
1. Sections from water to 50% and 70% alcohol.
2. Stain for up to 2 hours in saturated Sudan Black B in
70% ethanol.
3. Place in 70% alcohol for 5 seconds only to remove
excess surface dye. (Longer periods will remove the
color).
4. Immerse in 50% alcohol for I minute.
5. Wash in distilled water for 2 minutes.
6. Counterstain with Mayer's carmalum for 2 1/2 minutes.
7. Wash in distilled water for 2 minutes.
8. Mount in aqueous mounting medium.
Results: Lipids blue black & Nuclei Red
Sudan IV (Scharlach R) Stain for Lipids
Fixation: 10% Formalin
Sections: Frozen sections
Method: Collect 10 µ frozen sections in distilled water.
1. Place in 50% alcohol for 1 minute.
2. Place in 70% alcohol for 1 minute.
3. Immerse in Sudan IV or Scharlach R (Oil Red 0 may be
used) for 5-10 minutes.
4. Dip in 70% alcohol for 1minute (Longer periods will
remove the color).
5. Counterstain with Harris hematoxylin for 2 minutes.
6. Differentiate in 1% acid alcohol until only the nuclei are
stained blue/ black under microscopic control.
7. "Blue" in tap water for 5 minutes.
8. Rinse in distilled water.
9. Mount sections on to slide from distilled water to an
aqueous mounting medium.
Results: Lipids (mainly triglycerides) – red & Nuclei -
blue/black
NOTE:
- Scharlach R (Sudan IV) is the most commonly used
stain, producing a rapid and permanent coloration of
lipid. The addition of benzoic acid to the staining
solution materially intensifies the resulting color and
prevents deterioration. True fats stain intensely while
cholesterol stains less intensely.
Oil Red 0 Method
- closely related to Sudan dyes and was introduced in
1926 by French.
- popularized in 1943 by Lillie and Ashburn who
advocated its use as a 50 to 60% fresh aqueous dilution
of a saturated 99% isopropanol stock solution. Oil Red
0 stains neutral fats and lipofuscin well.
- The basis for staining lipids with an oil-soluble dye lies
in its increased solubility in fatty substances as opposed
to the dye solvents which are used in routine tissue
processing.
- The choice of solvent for this reaction is also critical,
since it must be able to extract excess dye without
dissolving the lipid to be stained
- propylene glycol: preferred solvent for this technique
Fixation: Fresh frozen or frozen sections post-fixed in
neutral buffered formalin
Sections: 5 µm sections mount on slides, air dry
Solutions:
• Dextrin Solution
Dextrin 1 gm
Distilled water 100 ml
• Oil Red O stock solution -
Oil Red O 0.5gm
Isopropanol 100ml
o Dissolve the dye in isopropanol using gentle heat in
water bath.
• Oil Red O working solution
Stock Oil Red O solution 30ml
Dextrin 20ml
o Allow to stand for a day or more. Stable or months,
filter before use.
Method:
1. Place slides directly into filtered 0.5% Oil Red O in
dextrin. Stain for 20 minutes, rinse with running water
briefly.
2. Counterstain with Gill II hematoxylin for 20-30
seconds. Rinse with water, blue, coverslip with aqueous
mounting media.
Results: Lipid red & Nuclei blue
Osmic Acid Stain for Fat
- Osmium tetroxide (osmic acid): is not a dye but is an
unstable oxide which is reduced to a permanent black
substance by unsaturated fats and fatty acids.
- used as a fixative for electron microscopy and in
histochemistry, and for demonstration of unsaturated
fats, with the disadvantage that other substances may
also be stained simultaneously.
- Lipids may be demonstrated by fixing the tissue in
chrome-osmium solutions or by using frozen section.
- Reduction of osmium by thiocarbohydrazide highly
enhances lipid labeling.
- After formol/ glutaraldehyde fixation much of the
lipid in the tissues is ‘bound’ and does not take up
osmium.
- Direct fixation with neutral osmium tetroxide is an
effective method for visualizing lipid for the electron
microscope, but the poor penetration of tissue by
osmium limits its use for light microscopy.
Fixation: 10% formalin
Section: Cryostat section
Method:
1. Collect frozen sections at l0 µ in distilled water.
2. Immerse in l % osmium tetroxide in the dark for 12-18
hours.
3. Wash in distilled water.
4. Wash thoroughly in tap water for 3 hours.
5. Counterstain if desired with l % safranin for l minute.
6. Rinse in distilled water.
7. Rinse in 70% alcohol.
8. Mount on to slides.
9. Blot dry.
10. Dehydrate rapidly, clear and mount.
Results: Nuclei yellow-orange & Fats black
Nile Blue Sulfate Method for Fats
- a dye capable of differentiating two lipid classes
simultaneously by the action of its two components:
a. red oxazone: dissolves neutral lipids
b. blue oxazine: basic and reacts with phospholipids
and free fatty acids.
- can be used as a preliminary indicator of the type of lipid
present in the tissue section.
- Nile red is an excellent stain that is present as a minor
component of commercial preparations of the non-
fluorescent lipid stain Nile blue.
o It is intensely fluorescent and it can serve as a
sensitive vital stain for the detection of cytoplasmic
lipid droplets.
o provides resolution of cytoplasmic lipid droplets in
tissues equal to that obtained with the non-
fluorescent dye Oil red 0
Histochemical Methods
- involve chemical reactions with specific groups,
radicals or bonds in the lipid molecule
➢ Free Fatty Acids
- bind heavy metal ions such as copper to form soaps
which can then be stained with Weigert's lithium
hematoxylin, dimethylaminobenzidine rhodamine, or
rubeanic acid.
- Calcium and iron deposits will also bind with copper,
but can be distinguished from lipid by their persistence
in a de-lipidized control section.
- They can be extracted with either 1% hydrochloric acid
(for calcium) or 5% oxalic acid (for iron salts).
➢ Cholesterol
- ineffective unless cholesterol had been oxidized, either
chemically with ferric salts or by long exposure to
atmospheric oxygen.
- An enzymatic method introduced by Emeis et al in 1977
based on the production of H2O2 from free cholesterol
by cholesterol oxidase.
- It involves a two-stage procedure in which esterified
cholesterol is initially hydrolyzed to its free sterol by
means of a cholesterol esterase. Cholesterol is then
oxidized by a cholesterol oxidase to release hydrogen
peroxide, which reacts simultaneously with
diaminobenzidine to produce an insoluble brown
polymer at the site of cholesterol.
- Cholesterol esters can be demonstrated as cholesterol
after hydrolysis by cholesterol ester hydrolase and has
also been detected with the use of enzyme cholesterol
esterase.
➢ Cerebrosides
- The sulfate esters of cerebrosides (sulfatides) are
generally deposited in brain and other organs of patients
with sulfatide storage disease known as metachromatic
leukodystrophy.
- stained by the Periodic Acid Schiff (PAS) method,
designed to stain mucopolysaccharides, and can be
distinguished from glycogen by removal with diastase.
- Staining with cresyl violet produces a metachromatic
orange color on sulfatides, in contrast to the
orthochromatic purple color of other, less acidic myelin
lipids.
- Toluidine blue is a standard metachromatic dye for
acidic polymers, and imparts a yellow brown or purplish
color to sulfatide deposits.
- The section is dehydrated with acetone to eliminate the
metachromasia induced by less acidic groups.
➢ Gangliosides
- Neurons may have significant ganglioside storage
giving rise to cells suggestive of gangliosidoses, but
storage may not be obvious particularly in subtypes
without mental retardation.
- The stored gangliosides are PAS (+), sudanophilic (+),
and Luxol fast blue (+).
- present in storage diseases like Tay-Sach's disease and
Gm1 gangliosidosis are stained with conventional PAS
method.
- distinguished from other glycolipids by their
constituents, neuraminic acid and sialic acid.
- A modified PAS method reduces the concentration of
the oxidizing agent (periodate) from 1 to 0.01% to stain
 sialo-groups that are oxidized more rapidly than other
sugar glycoside.
- This modified PAS stain has been used to demonstrate
gangliosides (the only glycolipids that contain sialic
acids) within neurons in Tay-Sach's disease.